1. Field of the Invention
The present invention relates to a novel class of trioxane dimers which demonstrate potent and potentially therapeutically valuable antiproliferative and antitumor activities.
2. Description of the State of Art
Artemisia annua L., also known as qing hao or sweet wormwood, is a pervasive weed that has been used for many centuries in Chinese traditional medicine as a treatment for fever and malaria. Its earliest mention, for use in hemorrhoids, occurs in the Recipes for 52Kinds of Diseases found in the Mawangdui Han dynasty tomb dating from 168 B.C. Nearly, five hundred years later Ge Hong wrote the Zhou Hou Bei Ji Fang (Handbook of Prescriptions for Emergency Treatments) in which he advised that a water extract of qing hao was effective at reducing fevers. In 1596, Li Shizhen, the famous herbalist, wrote that chills and fever of malaria can be combatted by qing hao preparations. Finally, in 1971, Chinese chemists isolated from the leafy portions of the plant the substance responsible for its reputed medicinal action. This crystalline compound, called qinghaosu, also referred to as QHS or artemisinin, is a sesquiterpene lactone with an internal peroxide linkage.
Artemisinin (3, 6, 9-trimethyl-9, 10b-epi-dioxyperhydropyranol [4, 3, 2-jk] benzoxepin-2-one) is a member of the amorphane subgroup of cadinenes and has the following structure (I) ##STR2## Artemisinin or QHS was the subject of a 1979 study conducted by the Qinghaosu Antimalarial Coordinating Research Group involving the treatment of 2099 cases of malaria (Plasmodium vivax and Plasmodium falciparum in a ratio of about 3:1) with different dosage forms of QHS, leading to the clinical cure of all patients. See, Qinghaosu Antimalarial Coordinating Research Group, Chin. Med. J., 92:811 (1979). Since that time QHS has been used successfully in several thousand malaria patients throughout the world including those infected with both chloroquine-sensitive and chloroquine-resistant strains of P. falciparum. Assay of QHS against P. falciparum, in vitro, revealed that its potency is comparable to that of chloroquine in two Hanian strains (Z. Ye, et al., J. Trad. Chin. Med., 3:95 (1983)) and of mefloquine in the Camp (cholorquine-susceptible) and Smith (chloroquine-resistant) strains, D. L. Klayman, et al., J. Nat. Prod., 47:715 (1984).
Although QHS is effective at suppressing the parasitemias of P. vivax and P. falciparum, the problems encountered with recrudescence, and the compound's insolubility in water, led scientists to modify QHS chemically, a difficult task because of the chemical reactivity of the peroxide linkage which is an essential moiety for antimalarial activity.
Reduction of QHS in the presence of sodium borohydride results in the production of dihydroartemesinin (II-1) or DHQHS, (illustrated in structure II below), in which the lactone group is converted to a lactol (hemiacetal) function, with properties similar to QHS. QHS in methanol is reduced with sodium borohydride to an equilibrium mixture of .alpha.- and .beta.-isomers of dihydroartemisinin. The yield under controlled conditions is 79% (QHS, 0.85M with NaBH.sub.4 6.multidot.34M. 7.multidot.5 equivalents in methanol, 12 L at 0-5.degree. C. for about 3 hours followed by quenching with acetic acid to neutrality at 0-5.degree. C. and dilution with water to precipitate dihydroartemisinin), A. Brossi, et al., Journal of Medicinal Chemistry, 31:645-650 (1988). Using DHQHS as a starting compound a large number of other derivatives, such as, ##STR3## artemether (compound II-2), arteether (II-3), sodium artesunate (II-4), artelinic acid (II-5), sodium artelinate (II-6), DHQHS condensation by-product (II-7) and the olefinic compound, structure III, ##STR4## have been produced.
Artemether (II-2) is produced by reacting .beta.-DHQHS with boron trifluoride (BF.sub.3) etherate or HCl in methanol:benzene (1:2) at room temperature. A mixture of .beta.- and .alpha.-artemether (70:30) is obtained, from which the former is isolated by column chromatography and recrystallized from hexane or methanol, R. Hynes, Transactions of the Royal Society of Tropical Medicines and Hygiene, 88(1): S1/23-S1/26 (1994). For arteether (II-3), (Brossi, et al., 1988), the .alpha.-isomer is equilibrated (epimerized) to the .beta.-isomer in ethanol:benzene mixture containing BF.sub.3 etherate. Treatment of DHQHS with an unspecified dehydrating agent yields both the olefinic compound, (III), and the DHQHS condensation by-product (II-7), formed on addition of DHQHS to (III), M. Cao, et al., Chem. Abstr., 100:34720k (1984). Until recently, the secondary hydroxy group in DHQHS (II-1) provided the only site in an active QHS related compound that had been used for derivatization. See B. Venugopalan "Synthesis of a Novel Ring Contracted Artemisinin Derivative," Bioorganic & Medicinal Chemistry Letters, 4(5):751-752 (1994).
The potency of various QHS-derivatives in comparison to QHS as a function of the concentration at which the parasitemia is 90 percent suppressed (SD.sub.90) was reported by D. L. Klayman, "Qinghaosu (Artemisinin): An Antimalarial Drug from China, " Science 228:1049-1055 (1985). Dr. Klayman reported that the olefinic compound III, is inactive against P. berghei-infected mice, whereas, the DHQHS condensation by-product (II-7), has an SD.sub.90 of 10 mg/Kg, in P. berghei-infected mice. Thus, the DHQHS ether dimer proved to be less potent than QHS, which has an SD.sub.90 of 6.20 mg/Kg. Following, in order of their overall antimalarial efficacy, are the three types of derivatives of DHQHS (II-1) that have been produced: (QHS)&lt;ethers (II, R=alkyl)&lt;esters [II, R.dbd.C(.dbd.O)-alkyl or -aryl]&lt;carbonates [II, R.dbd.C(.dbd.O)O-alkyl or -aryl].
Other rational designs of structurally simpler analogs of artemisinin has led to snythesis of various trioxanes, some of which possess excellent antinalarial activity. Posner, G. H., et al., reported the chemistry and biology of a series of new structurally simple, easily prepared, racemic 1,2,4-trioxanes (disclosed in U.S. Pat. No. 5,225,437 and incorporated herein by reference) that are tricyclic (lacking the lactone ring present in tetracyclic artemisinin I) and that are derivatives of trioxane alcohol IV ##STR5## having the relative stereochemistry shown above. Especially attractive features of trioxane alcohol IV are the following: (1) its straightforward and easy preparation from cheap and readily available starting materials, (2) its availability on gram scale, and (3) its easy one-step conversion, using standard chemical transformations, into alcohol derivatives such as esters and ethers, without destruction of the crucial trioxane framework. See, Posner, G. H., et al., J. Med. Chem., 35:2459-2467 (1992), incorporated herein by reference.
Over the past twenty years only a few drugs isolated from higher plants have yielded clinical agents, the outstanding examples being vinblastine and vincristine from the Madagascan periwinkle, Catharanthus roseus, etoposide, the semi-synthetic lignan, from May-apple Podophyllum peltatum and the diterpenoid taxol, commonly referred to as paclitaxel, from the Pacific yew, Taxus brevifolia. Of these agents, paclitaxel is the most exciting, recently receiving approval by the Food and Drug Administration for the treatment of refractory ovarian cancer. Since the isolation of QHS, there has been a concerted effort by investigators to study other therapeutic applications of QHS and its derivatives.
National Institutes of Health reported that QHS is inactive against P388 leukemia. See NCI Report on NSC 369397 (tested on Oct. 25, 1983). Later anticancer studies that have been conducted on cell line panels consisting of 60 lines organized into nine, disease-related subpanels including leukemia, non-small-cell lung cancer, colon, CNS, melanoma, ovarian, renal, prostate and breast cancers, further confirm that QHS displays very little anticancer activity. A series of artemisinin-related endoperoxides were tested for cytoxicity to Ehrlich ascites tumor (EAT) cells using the microculture tetrazolum (MTT) assay, H. J. Woerdenbag, et al. "Cytotoxicity of Artemisinin-Related Endoperoxides to Ehrlich Ascites Tumor Cells," Journal of Natural Products, 56(6):849-856 (1993). The MTT assay, used to test the artemisinin-related endoperoxides for cytoxicity, is based on the metabolic reduction of soluble tetrazolium salts into insoluble colored formazan products by mitochondrial dehydrogenase activity of the tumor cells. As parameters for cytoxicity, the IC.sub.50 and IC80 values, the drug concentrations causing respectively 50% and 80% growth inhibition of the tumor cells, were used. QHS (I), had an IC.sub.50 value of 29.8 .mu.M. Derivatives of DHQHS (II-1) being developed as antimalarial drugs (artemether (II-2), arteether (III-3), sodium artesunate (II-4), artelinic acid (II-5) and sodium artelinate (II-6)), exhibited a somewhat more potent cytoxicity. Their IC.sub.50 values ranged from 12.2 .mu.M to 19.9 .mu.M. The DHQHS condensation by-product (II-7), disclosed previously by M. Cao, et al., 1984, was the most potent cytotoxic agent, its IC.sub.50 being 1.4 .mu.M. At this drug concentration the condensation by-product (II-7), is approximately twenty-two times more cytoxic than QHS and sixty times more cytotoxic than DHQHS (II-1), the parent compound.
There is still a need, therefore, for developing structural analogs of QHS having antiproliferative and antitumor agents that have potency equivalent or greater than known anticancer antiproliferative and antitumor agents.